| Project/Area Number |
17K08549
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Multi-year Fund |
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| Section | 一般 |
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| Research Field |
General physiology
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| Research Institution | Tokyo Medical University |
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Principal Investigator |
Inoue Hana 東京医科大学, 医学部, 講師 (20390700)
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| Co-Investigator(Kenkyū-buntansha) |
村山 尚 順天堂大学, 医学部, 准教授 (10230012)
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| Project Period (FY) |
2017-04-01 – 2020-03-31
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| Project Status |
Completed (Fiscal Year 2019)
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| Budget Amount *help |
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2019: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2018: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2017: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
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| Keywords | TRPM7 / 平面膜 / キナーゼドメイン / 分子内相互作用 / 酸化ストレス / チャネル / シングルチャネル |
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| Outline of Final Research Achievements |
To understand the molecular mechanisms underlying regulations of TRPM7 channel activity, the single channel current of purified TRPM7 was recorded in planer lipid bilayers. The Streptavidin-Binding Peptide (SBP)-tagged TRPM7 protein was overexpressed in HEK293 cells by baculovirus-mediated transfection, and was purified by Strep-Tactin column. The purified TRPM7 protein was incorporated into lipid bilayers consisting of a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine:1-palmitoil-2-dioleoyl-sn-glycero-3-phosphoethanolamine at 3:1 ratio. The slope conductance and the open probability of TRPM7 wild type (TRPM7-wt) was 38 pS and 0.75 at +100 mV. Whereas, the conductance of TRPM7 lacking the kinase domain (TRPM7-dK) exhibited ~1.5 times higher than TRPM7-wt (56 at +100 mV). Moreover, the inhibition of TRPM7 by intracellular Mg2+ was augmented in TRPM7-dK. It was indicated that TRPM7 kinase domain regulates single channel conductance as well as intracellular Mg2+ sensitivity.
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| Academic Significance and Societal Importance of the Research Achievements |
TRPM7はノックアウトすると胎生致死であり、ヒトの原因不明の死産でもTRPM7変異が報告されており、個体の発生・生存に重要な分子であるといえる。TRPM7チャネルの立体構造は、キナーゼドメインを欠損したTRPM7-deltaKおよび膜貫通領域（チャネルドメイン）を持たないキナーゼドメインについてそれぞれ解かれている。本研究ではキナーゼドメインが細胞質マグネシウムイオンに対する感受性を制御すること、シングルチャネルコンダクタンスを減少させることを明らかにした。すなわち、全長TRPM7チャネルの構造は、TRPM7-deltaKと異なることが機能的に示唆された。
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